PL214839B1 - Using a factor for the preparation of a medicament for the treatment of glaucoma - Google Patents

Abstract

The present invention provides methods and kits for treating glaucoma. More particularly, the present invention provides for the use of an agent in the manufacture of a medicament for the treatment of glaucoma, wherein the agent is selected from the group consisting of: a chordin antagonist; a gremlin antagonist; and a follistatin antagonist and a topical ocular formulation suitable for the treatment of glaucoma and comprising from 0.01 % to 2.0% (wt. %) of an agent selected from the group comprising: a chordin antagonist; a gremlin antagonist; and a follistatin antagonist.

Description

The invention relates to the use of a gremlin antagonist in the manufacture of a medicament for the treatment of glaucoma.

"Glaucoma" is a group of eye disorders that are the leading cause of irreversible blindness in the United States and other developed societies. Primary open-angle glaucoma ("POAG"), the most common form of glaucoma, is characterized by the degeneration of the corneal scleral trabeculae, disrupting the normal ability of the aqueous humor to leave the eye without constricting the space (e.g., "angle") between the iris and the cornea (Vaughan, D. et al. (1992)). The hallmark of this disorder in this condition is increased intraocular pressure ("IOP"), leading to gradual loss of vision and blindness, if not treated properly and in a timely manner.

It is estimated that the disease affects 0.4% -33% of all adults over the age of 40 (Leske, M. C. et al. (1986); Bengtsson, B. (1989); Strong, N. P. (1992)). In addition, the incidence of the disease increases with age to more than 6% in those 75 years of age and older (Strong, N. P., (1992)).

Since elevated IOP is an easily measurable feature of glaucoma, diagnosis is mainly made by measuring intraocular pressure (tonometry) (Strong, N. P. (1992); Greve, M. et al. (1993)). Unfortunately, since glaucoma and normal pressure ranges overlap, such methods are of limited value unless multiple readings are performed (Hitchings, RA, (1993); Tuck, MW et al. (1993); Vaughan, D. and et al. (1992); Vernon, SA, (1993)). Therefore, additional methods are often performed to improve the accuracy of diagnosis, such as direct examination of the optic disc and determining the degree of visual field loss in a patient (Greve, M. et al., (1993)).

Glaucoma affects three separate tissues in the eye. The elevated IOP associated with POAG is caused by morphological and biochemical changes in the trabecular (TM) weaving of the tissue located at the angle between the iris and the cornea. Most of the eye's nourishing fluid comes out of the front of the eye through the TM. The gradual loss of TM cells and the formation of extracellular deposits in TM of glaucoma eyes leads to an increased resistance to water outflow (LutjenDrecoll and Rohen 1996; Rohen 1983; Rohen et al. 1993; Grierson and Calthorpe 1988), thus increasing IOP. Elevated IOP as well as other factors such as ischemia cause degenerative changes in the optic nerve head (ONH) leading to the gradual formation of a depression in the ONH (Varma and Minckler 1996; Hernandez and Gong 1996; Hernandez et al. 1990; Hernandez and Pena 1997 ; Morrison et al. 1990) and loss of retinal ganglion cells (Quigley et al. 2000; Quigley 1999; Quigley et al. 1995; Kerrigan et al. 1997) and axons. The detailed molecular mechanisms responsible for glaucoma-related damage to TM, ONH, and ganglion cells are unknown.

Current treatments for glaucoma are aimed at lowering IOP, a major risk factor for the development and progression of glaucoma. These therapies lower IOP but do not directly target pathogenic mechanisms and the disease continues to develop. At least half of glaucoma patients go undiagnosed, and by the time patients are diagnosed with glaucoma, they have lost about 40% of their retinal ganglion cells. Thus, methods for the early detection and diagnosis of glaucoma are needed.

Given the importance of glaucoma, and at least partially the mismatch of prior methods of diagnosis, it would be desirable to have an improved, more accurate, method of diagnosing glaucoma in the early stages. In addition, therapeutic agents that target glaucoma pathogenesis mechanisms are desirable.

The present invention overcomes these and other prior art limitations. The invention relates to the use of a gremlin antagonist in the manufacture of a medicament for the treatment of glaucoma, wherein the gremlin antagonist is an antibody.

Figures

Fig. 1. Signal pathway of bone morphogenesis proteins.

Bone morphogenetic protein (BMP) dimers bind to the membrane complex consisting of BMP receptors 1 and 2, which are serine / threonine kinases. Regulatory Smad (Smadl / Smad5) becomes phosphorylated and binds to co-Smad (Smad 4). This produced Smad complex enters the nucleus where it binds / interacts with transcription factors (TF) and regulates

Gene expression. BMP associated proteins act as BMP antagonists by binding to BMP and preventing BMP from interacting with BMP receptors. Gremlin is also an antagonist protein.

Figure 2. mRNA expression of BMP related proteins in human TM cells.

Agarose gel stained with ethidium bromide with PCR products from cDNA samples derived from RT-PCR analysis of human TM cells (lanes 1-5). L = base pair markers. C = PCR negative control lane. β-actin was used as internal positive control in RT-PCR.

Figure 3. Expression of BMP-related protein mRNA in human ethmoid cells and ONH astrocytes.

Agarose gel stained with ethidium bromide with PCR products from cDNA samples derived from RT-PCR analysis of ethmoid lamina (LC) cells (lanes 1-7) and ONH astrocytes (ONA) (lanes 8-11). L = base pair markers. C = PCR negative control lane. β-actin was used as internal positive control in RT-PCR.

Figure 4. Shows the increased expression of gremlin-BMP antagonist (CKTSF1B1) in glaucoma TM cells.

Gene expression was determined using the Affymetrix gene microarray (Affymetrix gene chip

U133A).

Trabecular meshwork is believed to play an important role in the normal flow of the aqueous humor and is believed to be the major site of drainage resistance in glaucoma eyes. Human trabecular meshwork (HTM) cells are specialized cells that line the drainage tubes through which the aqueous humor exits the eye. The altered synthetic function of these cells may be involved in the pathogenesis of POAG, steroid glaucoma and other types of glaucoma.

Despite years of intense research, the detailed molecular processes responsible for eye damage in glaucoma remain unknown. Recent studies have suggested that growth factors may be important in maintaining normal homeostasis in glaucoma-related eye tissues and changes in growth factors / growth factor receptors may play a role in the pathogenesis of glaucoma. Growth factors are a very large family of polypeptides that control cell growth and differentiation. These molecules have a variety of cell-specific effects on gene expression, extracellular matrix composition and deposition, cytoskeleton organization, and regulation of cellular functions. TM produces a wide variety of growth factors, growth factor receptors (Tripathi et al. 1993a; Tripathi et al. 1993b; Tripathi et al. 1994a; Tripathi et al. 1994b; Wordinger et al. 1998; Wordinger et al. 1999) as well as neurotrophins / neurotrophic factors and their receptors (Liu et al. 2001; Wordinger et al. 2000). ONH astrocytes and ethmoid cells, two types of optic head cells, express growth factors, neurotrophins and their receptors (Lambert et al. 2001; Pena et al. 1999). The aqueous humor of the eye also contains a variety of growth factors including FGF2, EGF, TGF3, HGF (Tripathi et al. 1996; Tripathi et al. 1991; Tripathi et al. 1992; Hu and Ritch 2001) as well as neurotrophins (Chundru et al. 2000) . Elevated levels of TGF3-2 and HGF in the aqueous humor have been reported in POAG patients (Tripathi et al. 1994c; Inatani et al. 2001; Picht et al. 2001). Growth factors may be involved in glaucoma by altering the normal development and / or function of TM and ONH.

The present invention is based in part on the finding that bone morphogenetic proteins (BMPs) not only induce bone and cartilage formation, but are multifunctional cytokines having a wide range of effects on multiple cell types (Hogan 1996; Reddi 1997) and are both expressed by human weave trabecular (HTM) and optic head (ONH) cells (Wordinger et al. 2002). BMPs are members of the TGF3 superfamily and there are approximately 15-20 BMP genes in humans, 3 BMP receptors, and a number of BMP-related proteins that act as BMP antagonists (Ymashita et al. 1996). BMPs signal through a receptor complex consisting of BMPR-I and BMPR-II. Members of the TGF3 and TGF3R superfamily (Agarwal et al. 1997; Lambert et al. 1997) and GDNF and GDNFR (Wordinger et al. 1999; Liu et al. 1999) have been reported to be expressed by both HTM and ONH cells.

BMP and BMP receptors are expressed in eye tissues (Obata et al. 1999; You et al. 1999), but the reports so far have focused on eye development. The function of BMP is important to eye development because targeted disruption of the genes encoding BMP in mice leads to severe developmental disorders in the retina and lens (Jena et al. 1997; Luo et al. 1995; Dudley et al. 1995). BMP-2, BMP-4 and BMP-7 are involved in the development of the retina and lens (Jena et al. 1997; Furuta and Hogan 1998; Reddi 2000; Trousse et al. 2001). BMP-6 and BMP-7 also appear to play a role in protecting neurons from damage caused by hypoglycemia or hypoxia (Nonner et al. 2001;

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Liu et al. 2001) and BMP2 has been shown to increase the expression of neurotrophin in ganglion cells (Zhang et al. 1998). Heterozygous knock-out mice with haploid Bmp4 deficiency show ocular phenotypes including posterior ventricular dysgenesis, elevated IOP, and abnormalities in the optic nerve (Chang et al. 2001). Very limited information has been published regarding the role of BMP in the human eye after birth.

Mohan et al. (1998) reported that BMP-2 and BMP-4 and BMP receptors are expressed in adult corneal cells and suggested that the function of BMP may involve division and apoptosis of the corneal keratocytes. You et al. (1999) reviewed this study and also reported expression of BMP-3, BMP-5 and BMP-7 in ex vivo and cultured corneal epithelium and stroma cells. They reported that the transcription level for BMP was higher in the stroma, while the level for the receptors was higher in cultured corneal endothelial cells.

Using RT-PCR, the present inventors also revealed the mRNA of the BMP binding proteins gremlin, chordin, follistatin and bambi in HTM cell lines, ethmoid lamina (LC) and ONH astrocytes and tissues (Wordinger et al. 2002). The present inventors have further found that HTM and ONH cells express BMP-2, BMP-4, BMP-5 and BMP-7 proteins.

Glaucoma can be diagnosed by characterizing genetic changes in the genes of members of the BMP signaling family. As accepted herein, the expressions "gene member of the bone morphogenetic protein family" and "BMP signaling family" refer to all BMPs, BMP receptors, and associated proteins. The term "genetic alteration is well known to those skilled in the art. There are numerous examples of diseases associated with genetic alterations in specific genes (for example, see Cummings 1997; Strachan, et al. 1996; Jorde, et al. 1999). Genetic changes in a particular gene (e.g., BMP) can be determined by a variety of techniques well known to those skilled in the art, such as SSCP, DGGE, ASO, RFLP, heteroduplex analysis, CCM, PTT, and RNase digestion (see Birren et al. 1998).

Glaucoma can be caused by altered expression of one or more genes of the BMP family in the eye, leading to elevated IOP and / or neuropathies characteristic of glaucoma. "Altered BMP gene expression" means expression of a product of that gene which differs from the normal. The term can also refer to changes in the sequence of a gene or protein. The normal BMP gene has been well characterized (see above) and expression of BMP in various tissues including TM and ONH has been described. Genetic changes in the coding region of genes from the BMP family can alter the function of these proteins. Genetic changes outside the coding region can also lead to glaucoma.

As is well known to those skilled in the art, "changes outside" the coding region of a particular gene are important in regulating gene expression. For example, the region upstream (5 ') of the coding region of most genes is known as a promoter region that "promotes" and regulates expression of that gene. The promoter region contains numerous nucleotide sequences recognized by a variety of transcription factors and DNA binding proteins that are responsible for the activation or repression of gene expression. The regions downstream (3 ') of the gene can determine the polyadenylation of the gene product, thus regulating RNA processing and translation of the gene product.

Altered expression of the BMP genes or mutations in the gene sequence that indicate glaucoma can be detected using techniques well known to those skilled in the art.

The therapeutic agent for treating glaucoma is an antibody gremlin antagonist.

The medicament prepared in accordance with the use of the invention can be administered directly to the eye (e.g., topical eye drops or ointments; drug slowly releasing therapeutic systems (inserts) in the conjunctival sac or implanted next to the sclera or inside the eye; periocular, conjunctival, or under the Tenon's capsule injections. , intraventricular, intravitreal) or parenterally (e.g., oral, intravenous, subcutaneous, intramuscular, transdermal, etc.) using methods well known to those skilled in the art. It has also been found that formulations containing a gremlin antagonist can be formulated as therapeutic systems (inserts) placed inside the eye.

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T a b e l a 1

BMP family members expressed in human TMs and ONHs

A representative of the BMP family Trabecular weave The head of the optic nerve BMP-2 + + BMP-4 + + BMP-5 + + BMP-7 + + BMPR-1A + + BMPR-1B + + BMPR-II + + Chordina + + Gremlin + + Follicular + + Bambi + + Noggin - - CER-1 - -

An example of the composition of a formulation according to the invention is shown below.

Topical eye composition% by weight

Gremlin antagonist 0.01-2

HPMC 0.5

Sodium chloride 0.8

BAC 0.01

EDTA 0.01

NaOH / HCl to pH 7.4

Purified water up to 100 ml.

The following references, insofar as they provide exemplary methodological or other additional details to those provided herein, are specifically incorporated herein by reference.

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Claims (1)

The use of a gremlin antagonist in the manufacture of a medicament for the treatment of glaucoma, wherein the gremlin antagonist is an antibody.